The formation of perfectly aligned myofibrils in cardiac muscle is a dramatic example of supramolecular assembly in eukaryotic cells; the mechanisms by which this occur are still incompletely understood. The long term objective of this research is to identify the molecular components and mechanisms that regulate sarcomeric protein interactions during de novo myofibril assembly: properties required for efficient contractile activity. The goal of this proposal is to define the functional roles of erythrocyte tropomodulin (E-Tmod) and its specific molecular interactions with actin, tropomyosin and a novel cardiac nebulin in regulation of thin filament assembly, length and dynamics during distinct stages of de novo myofibril assembly. We hypothesize that E-Tmod is a multifunctional protein and that its separate domains serve distinct physiological roles during particular stages of heart development. A combination of advanced cell and molecular approaches, in conjunction with two unique culture systems, chick precardiac explants and murine E-Tmod knockout embryonic stem (ES) cells, will be utilized. The aims are: 1) Define the minimal functional binding sites necessary for the interaction of E-Tmod with tropomyosin, and with cardiac nebulin; 2) Determine the mechanistic relationship between E-Tmod's assembly at thin filament pointed ends with its specific ligand interactions, its dynamics, actin isoform expression patterns and dynamics, and regulation of thin filament lengths during distinct stages of myofibril assembly in live differentiating cardiac myocytes. Also, GFP-E-Tmod fragments and mutants with their individual, or combination of, binding sites specifically abrogated will be analyzed for their ability to target to thin filament ends in real time; 3) Define the functional consequences of ectopic expression of mutated forms of E-Tmod, and its binding partners, to selectively inhibit each interaction during distinct stages of myofibril assembly in both chick precardic explants and E-Tmod (-/-) ES cells. Additionally, other T-mod isoforms will be expressed in the E-Tmod (-/-) ES cells to determine their roles in the null background. Deciphering the mechanisms of thin filament dynamics and determining the roles of key regulatory proteins such as E-Tmod are pivotal for understanding the molecular bases for myopathies seen in various types of heart disease, including familial hypertrophic cardiomyopathies.